High-efficiency transfection of biological cells using sonoporation

ABSTRACT

A method is provided for achieving transfection of host cells using sonoporation. An acoustic radiation generator is positioned in acoustic coupling relationship with respect to a reservoir containing host cells to be transfected, exogenous material to be incorporated into the host cells, and a cell-compatible fluid medium. The acoustic radiation generator is activated to generate acoustic radiation and direct the acoustic radiation into the reservoir in a manner effective to enable transfection of the host cells with the exogenous material.

CROSS-REFERENCE TO EARLIER APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/706,524, filed Sep. 15, 2017, which claims priority under 35 USC119(e)(1) to provisional U.S. Patent Application Ser. No. 62/439,458,filed Dec. 27, 2016, and to provisional U.S. Patent Application Ser. No.62/395,363, filed Sep. 15, 2016, the disclosures of which areincorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to biotechnology, and moreparticularly relates to methods and systems for the efficienttransfection of biological cells. The invention finds utility in thefields of biochemistry and medicine, including cellular research anddrug discovery.

BACKGROUND

Transfection refers to the incorporation of foreign material into hostcells, either bacterial or mammalian. In the realm of biotechnology,transfection has become a critically important tool used to introduceforeign DNA or RNA into cells in order to produce genetically modifiedcells. Transfection may be either stable or transient. In stabletransfection, the introduced genetic material is delivered to the hostcell nucleus by passage through the cell and nuclear membranes, andbecomes integrated into the host genome; every daughter cell has theadded material. In transient transfection (also referred to as“transformation”), by contrast, the nucleic acid is inserted into thehost cell but does not become integrated into its genome. As a result,the foreign genetic material is expressed temporarily but does not passto future generations of the transfected cell. Accordingly, it will beappreciated that stable transfection is necessary for large-scaleprotein production, gene therapy, drug discovery, compound screening,and extended research. The development of stable cell lines, however, iscomplex, time- and labor-consuming, and costly.

There are various methods of introducing foreign genetic material into aeukaryotic host cell, including biologically, chemically, and physicallymediated techniques. The most commonly used transfection method inresearch is biological, and involves the use of a virus as carrier.Adenoviral, lentiviral, and oncoretroviral vectors have been usedextensively for gene delivery in mammalian cell culture and in vivo.Virus-mediated transfection, or viral “transduction,” is efficient andrelatively straightforward to use, even with cell types that aredifficult to transfect. There are significant drawbacks, however,including the immunogenicity and cytotoxicity of the selected virus aswell as the difficulty and time involved in producing viral vectors.Lentiviral vectors, for instance, are also biohazardous to the user andrequire Biosafety Level 2 (BSL-2, as established by the U.S. Centers forDisease Control and Prevention) or Enhanced BSL-2 (BSL-2+) workingconditions.

Chemical transfection methods are widely used, and they were the firstto be used to introduce foreign genes into mammalian host cells.Chemical methods commonly used include, without limitation, thefollowing: calcium phosphate combined with a buffered saline/phosphatesolution; cationic polymers such as a conjugate of diethylethanolamineand dextran (or “DEAE-dextran) or polyethyleneimine; cationic lipidformulations such as that commercially available under the tradenameLipofectamine® (additional cationic lipid formulations are described inthe pertinent texts and literature, e.g., by Felgner et al. (1994) J.Biol. Chem. 269(4):2550-61); and activated dendrimers, such aspolyamidoamine dendrimers (see Hudde et al. (1999) Gene Therapy6(5):939-943). Chemical transfection efficiency varies depending on celltype, genetic material/chemical transfection agent ratio, solution pH,and other conditions. While chemical transfection methods are notassociated with the potential immunogenicity and cytotoxicity of viraltransfection agents, they generally exhibit poor transfectionefficiency. Furthermore, many of the aforementioned chemicaltransfection reagents can be used with only a very small number of celllines, those that are robust and not particularly sensitive, e.g., HeLaor HEK-293 cells.

Physical transfection methods are more recent than either viral orchemical transfection, and include techniques such as electroporation,laser-based transfection, biolistic particle delivery, cell squeezing,and direct micro-injection. While these methods have been established toachieve transfection, there are numerous associated problems, includingthe potential for extensive physical damage to samples.

In order to overcome some of the problems encountered with theaforementioned methods, there has been some effort put into using thetechnique of “sonoporation” to achieve transfection. Sonoporationinvolves the use of ultrasound, or acoustic energy, to induce atransient change in cell membrane permeability sufficient to allow theuptake of macromolecules by a host cell, where those macromoleculeswould otherwise not pass through the cell membrane. The work done todate in this area has focused on the use of an ultrasound contrast agent(UCA). Most UCAs are microbubbles filled with a buoyant gas, and aredesigned for use in medical ultrasound testing to increase acousticreflectivity via backscattering. It has been proposed that UCAs be usedin sonoporation by undergoing cavitation in the proximity of a hostcell, such that the UCA first expands, but then rapidly contracts orcollapses, generating microstreams or shock waves that apply shearstress to cell membranes, temporarily or permanently rupturing thosemembranes. More recently, it has been suggested that sonoporation canoccur using UCAs with acoustic energies below that which would causecavitation. See, e.g., Forbes et al. (2008) Ultrasound in Med. & Biol.34(12):2009-2018. The process has not been implemented on a largerscale, however, for a variety of reasons, including the fact that in aliquid medium, UCAs will rise to the liquid surface, while the hostcells will gravitate downward. This is problematic, since transfectionvia sonoporation requires that the buoyant microbubbles and the hostcells be adjacent when acoustic energy is applied. Another issue isefficiency: to date, there has been no report of a sonoporation-basedtransfection method in which the number of successfully transfected hostcells is maximized while cell death is minimized. Furthermore, likeother transfection techniques, sonoporation, to date, has beenineffective in transfecting for cells that are difficult to transfect,e.g., primary cells, particularly stem cells.

An ideal transfection method would do the following:

Maximize the fraction of host cells that are transfected whileminimizing cell death;

Allow for the successful transfection of a variety of cell types,including cells that are typically resistant to transfection;

Enable transfection of non-mammalian cells as well as mammalian cells;

Enable transfection of confluent as well as non-confluent cells;

Allow for transfection of cells with nucleic acids such as DNA, RNA,small interfering RNA (siRNA/RNAi), micro RNA (miRNA), and DNA plasmids;

Allow for transfection of cells with other types of exogenous material,including proteins and small molecules;

Work with nucleic acid sequences and associated proteins selected formodification of target cell function and machinery for execution of geneexpression, including (a) ribonucleoproteins (RNPs) composed of Cas9protein and guide RNA and (b) CRISPR plasmid with associated promoters;

Involve straightforward implementation without requiring an inordinateamount of time or labor; and

As a result of speed and ease of implementation, be adaptable to use inhigh-throughput transfection.

SUMMARY OF THE INVENTION

Accordingly, the present invention addresses the above-discussed need inthe art and provides a sonoporation-based method for transfecting hostcells.

In one embodiment, the invention provides an acoustic method fortransfecting cells, the method comprising:

(a) providing a system that comprises (i) at least two reservoirs eachcontaining host cells and exogenous material to be introduced into thehost cells, and (ii) an acoustic radiation generator to generate anddirect acoustic radiation;

(b) acoustically coupling the acoustic radiation generator to a first ofthe reservoirs without simultaneously acoustically coupling the acousticradiation generator to any other of the reservoirs;

(c) activating the acoustic radiation generator to generate and directacoustic radiation into the first reservoir in a manner that inducessonoporation of the host cells, thereby facilitating introduction of theexogenous material into the sonoporated host cells;

(d) acoustically decoupling the acoustic radiation generator from thefirst reservoir;

(e) acoustically coupling the acoustic radiation generator to a secondof the reservoirs without simultaneously acoustically coupling theacoustic radiation generator to any other of the reservoirs; and

(f) repeating step (c) with respect to the second reservoir.

In one aspect of this embodiment, the at least two reservoirs arecontained within a plurality of reservoirs, and the method furtherincludes (g) acoustically decoupling the acoustic radiation generatorfrom the second reservoir and thereafter repeating steps (b) through (g)with respect to additional reservoirs. The reservoirs may be containedwithin an integrated multiple reservoir unit such as a microwell plate,such as a 96-well plate, a 384-well plate, a 1536-well plate, or thelike. The acoustic radiation directed into the reservoir, in one aspectof this embodiment, is focused acoustic radiation.

In another aspect of this embodiment, the method is carried out withinthe context of a high-throughput transfection system, in which hostcells in each of a plurality of multiple reservoirs are rapidlysonoporated in succession. This may mean a reservoir-to-reservoirtransition time of at most about 0.5 seconds, 0.1 seconds, or 0.001seconds. In a related aspect, the volume of fluid medium in eachreservoir may be in the range of about 0.5 μL to about 500 μL.

In another aspect of this embodiment, the manner for inducingsonoporation includes a means for imparting the the acoustic radiationgenerated to the host cells, generally a transfection excitationmaterial that comprises a plurality of acoustically activatable moietieswithin the fluid medium, such as acoustically activatable localizedfluid volumes. The localized fluid volumes may be gas-filledmicrobubbles, which may be conjugated to the host cells to facilitatetransfer of acoustic energy from the irradiated microbubbles to the hostcells.

In another embodiment, a method is provided for transfecting host cells,comprising:

(a) preparing a microbubble composition by suspending, in a fluid mediumcompatible with the host cells, a plurality of gas-filled microbubblessurface-functionalized with a first binding moiety;

(b) conjugating the microbubbles to antibodies specific for the hostcell type and functionalized with a second binding moiety that links tothe first binding moiety, by mixing the microbubbles with the antibodiesin the fluid medium, thereby creating microbubble-antibody conjugates;

(c) preparing loaded microbubble-antibody conjugates by mixing themicrobubble-antibody conjugates with an exogenous material to betransfected into the host cells;

(d) optionally diluting the loaded microbubble-antibody conjugates witha host cell-compatible fluid medium that provides a dilution having aloaded microbubble-antibody conjugate concentration effective tooptimize transfection efficiency;

(e) contacting host cells in a reservoir with the dilution;

(f) sonoporating the host cell-dilution mixture provided in (e) byirradiating the reservoir with acoustic radiation under conditions thatcause the microbubbles to resonate at a frequency within about 15% oftheir average resonance frequency or within about 15% of a harmonic oftheir average resonance frequency.

In another embodiment, the invention provides an acoustic method fortransfecting cells that comprises: acoustically coupling an acousticradiation generator to a reservoir that contains host cells, exogenousmaterial to be transfected into the host cells, and a fluid medium; andactivating the acoustic radiation generator to generate acousticradiation and direct the acoustic radiation into the reservoir in amanner that induces sonoporation of the host cells without resulting ina temperature increase in the fluid medium of greater than about 10° C.

In another embodiment, the invention provides an acoustic method fortransfecting cells that comprises: acoustically coupling an acousticradiation generator to a selected reservoir contained within an integralmultiple reservoir unit comprising at least 1536 reservoirs, theselected reservoir containing host cells, exogenous material to betransfected into the host cells, and a fluid medium; and activating theacoustic radiation generator to generate acoustic radiation and directthe acoustic radiation into the reservoir in a manner that inducessonoporation of the host cells, thereby facilitating incorporation ofthe exogenous material into the sonoporated host cells.

In another embodiment, the invention provides an acoustic method fortransfecting cells that comprises: acoustically coupling an acousticradiation generator to a reservoir that contains host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprised of a plurality ofacoustically activatable localized fluid volumes; and activating theacoustic radiation generator to generate acoustic radiation and directthe acoustic radiation into the reservoir in a manner that acousticallyactivates the localized fluid volumes so that they vibrate at afrequency that is within about 15% of the average resonance frequency ofthe localized fluid volumes or within about 15% of a harmonic of theaverage resonance frequency of the localized fluid volumes, therebyfacilitating incorporation of the exogenous material into host cells inthe proximity of the acoustically activated localized fluid volumes.

In another embodiment, the invention provides an acoustic method fortransfecting cells that comprises: acoustically coupling an acousticradiation generator to a reservoir that contains host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprised of a plurality ofacoustically activatable localized fluid volumes having a sizedistribution; and activating the acoustic radiation generator togenerate acoustic radiation and direct the acoustic radiation into thereservoir in a manner that acoustically activates the localized fluidvolumes having a size within about 15% of a selected size, therebyfacilitating incorporation of the exogenous material into host cells inthe proximity of the acoustically activated localized fluid volumes.

In a related embodiment, the invention provides an acoustic method fortransfecting cells that comprises: (a) acoustically coupling an acousticradiation generator to a reservoir that contains host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprised of a plurality ofacoustically activatable localized fluid volumes having a multimodalsize distribution; (b) activating the acoustic radiation generator togenerate acoustic radiation and direct the acoustic radiation into thereservoir in a manner that acoustically activates localized fluidvolumes having a size that is within about 15% of a first modal peak,whereby the acoustically activated localized fluid volumes transferacoustic energy to nearby host cells; (c) repeating step (b) toacoustically activate localized fluid volumes having a size that iswithin about 15% of a second modal peak; and (d) optionally repeatingstep (b) to acoustically activate localized fluid volumes having a sizethat is within about 15% of one or more additional modal peaks.

In an additional embodiment, the invention provides an acoustic methodfor transfecting cells that comprises: acoustically coupling an acousticradiation generator to a reservoir containing host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprised of a plurality ofacoustically activatable localized fluid volumes having a sizedistribution; and activating the acoustic radiation generator togenerate acoustic radiation having a selected frequency content anddirect the acoustic radiation generated into the reservoir in a mannerthat induces sonoporation of the host cells, wherein the frequencycontent of the acoustic radiation generated is selected to correlatewith the size distribution of the acoustically activatable localizedfluid volumes.

In a related embodiment, the invention provides an acoustic method fortransfecting cells that comprises: acoustically coupling an acousticradiation generator to a reservoir containing host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprised of a plurality ofacoustically activatable localized fluid volumes having a spatialdistribution within the reservoir; and activating the acoustic radiationgenerator to generate acoustic radiation having a selected frequencycontent and direct the acoustic radiation generated into the reservoirin a manner that induces sonoporation of the host cells, therebyfacilitating incorporation of the exogenous material into thesonoporated host cells, wherein the frequency content of the acousticradiation generated is selected to correlate with the spatialdistribution of the acoustically activatable localized fluid volumes.

In another embodiment, sonoporation is conducted using two transducersoperating in concert (preferably but not necessarily simultaneously) butat different frequencies, wherein one of the transducers is an annulartransducer is operably mounted around and enclosing a standardtransducer. In this embodiment, the annular transducer and the standardtransducer will generally operate at different frequencies. In oneaspect of this embodiment, the annular transducer may operate at afrequency selected to bring about sonoporation, while the standardtransducer can be operated at a frequency effective to result inacoustic ejection of sonoporated cells, e.g., into a reservoir, onto asubstrate, or to an analytical instrument for analysis. In anotheraspect of this embodiment, one of the two transducers primarilyfunctions to supply the acoustic energy for sonoporation and the othertransducer delivers acoustic energy to change the relative position ofthe microbubbles with respect to the host cells when microbubble-cellconjugation is not used.

In another embodiment, sonoporation is carried out by irradiating withmultiple acoustic tonebursts in succession, each having a differentacoustic frequency effective to sonoporate differently sizedmicrobubbles. The acoustic frequency of each of the multiple acoustictonebursts is typically in the range of about 1.5 MHz to about 5.0 MHz,more usually in the range of about 2.0 MHz to about 2.5 MHz.

In a further embodiment, an acoustic method for transfecting cells isprovided that comprises: acoustically coupling an acoustic radiationgenerator to a selected reservoir containing host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprising a plurality of acousticallyactivatable localized fluid volumes; and activating the acousticradiation generator to generate acoustic radiation and direct theacoustic radiation into the reservoir in a manner that acousticallyactivates the localized fluid volumes, thereby facilitatingincorporation of the exogenous material into host cells in the proximityof the acoustically activated localized fluid volumes, wherein theacoustic radiation generated is at an acoustic sonoporation pressureselected to ensure that at least 50% of the localized fluid volumesremain intact after irradiation. In one aspect of this embodiment, theacoustic sonoporation is in the range of about 50% to about 90% of theminimum acoustic pressure that would result in cavitation of thelocalized fluid volumes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a plot of forward versus side scatter height in the FACSanalysis described in Example 4.

FIG. 2 also derives from the FACS analysis described in Example 4, andindicates that the successfully transfected were detected in the FL1channel, while dead cells were detected in the FL3 channel.

FIG. 3 also derives from the FACS analysis described in Example 4, andseparately illustrates the results obtained for the positive controls.

FIG. 4 provides well-by-well results illustrating that the fraction oftransfected cells in Example 4 increased with both acoustic power andmicrobubble concentration.

FIG. 5 provides the results in graph form.

FIG. 6 shows, well by well, that the percentage of live cells remainingpost-sonoporation in Example 14 was near 100%, even at the highervoltage used, 1.5 V.

FIG. 7 illustrates the data obtained for the transfected cells and thenegative control (i.e., DPBS only, in the absence of microbubbles) inExample 4.

FIG. 8 shows the percentage of GFP-positive cells obtained for each offour plasmid concentrations in the CRISPR transfection experimentdescribed in Example 5.

FIG. 9 shows the average percentage of GFP-positive cells at each offour plasmid concentrations, with standard deviation error barsindicated.

FIG. 10 shows the percentage of dead cells for each of the four plasmidconcentrations, as described in Example 5.

FIG. 11 illustrates the results of the mismatch cleavage assay andanalysis for the CRISPR transfection experiment of Example 6, Run 1.

FIG. 12 illustrates the results of the mismatch cleavage assay andanalysis for the CRISPR transfection experiment of Example 6, Run 2.

FIG. 13 is a bar graph indicating the percentage cleavage resultsobtained for Example 6, Runs 1 and 2.

FIG. 14 provides fluorescence images obtained for the transfected hostcells in Example 6, Run 2, using an EVOS fluorescent microscope with anRFP light cube to detect the labeled tracrRNA.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Specific terminology of particularimportance to the description of the present invention is defined below.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, “a component” refers not only toa single component but also to a combination of two or more differentcomponents, and the like.

The terms “acoustic radiation” and “acoustic energy” are usedinterchangeably herein and refer to the emission and propagation ofenergy in the form of sound waves. As with other waveforms, acousticradiation may be focused using a focusing means, as discussed below.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like a lens,or by the spatial arrangement of acoustic energy sources to effectconvergence of acoustic energy at a focal point by constructive anddestructive interference. A focusing means may be as simple as a solidmember having a curved surface, or it may include complex structuressuch as those found in Fresnel lenses, which employ diffraction in orderto direct acoustic radiation. Suitable focusing means also includephased array methods as are known in the art and described, for example,in U.S. Pat. No. 5,798,779 to Nakayasu et al. and Amemiya et al. (1997)Proceedings of the 1997 IS&T NIP13 International Conference on DigitalPrinting Technologies, pp. 698-702.

The terms “acoustic coupling” and “acoustically coupled” used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two items are indirectly acoustically coupled, an “acousticcoupling medium” is needed to provide an intermediary through whichacoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid in a reservoir, by, for example,interposing an acoustic coupling medium between the ejector and thefluid to transfer acoustic radiation generated by the ejector throughthe acoustic coupling medium and into the fluid.

An “acoustically activatable” moiety is a moiety that is caused tovibrate at an ultrasonic frequency when irradiated with acoustic energyof a particular wavelength.

The term “reservoir” as used herein refers to a receptacle or chamberfor holding or containing a fluid. In its one of its simplest forms, areservoir consists of a solid surface having sufficient wettingproperties to hold a fluid merely due to contact between the fluid andthe surface. A reservoir may also be a well within a well plate, a tubeor other such container in a tube rack, and the like.

The term “array” as used herein refers to a two-dimensional arrangementof features, such as an arrangement of reservoirs, e.g., wells in a wellplate. Arrays are generally comprised of features regularly ordered in,for example, a rectilinear grid, parallel stripes, spirals, and thelike, but non-ordered arrays may be advantageously used as well. Anarray differs from a pattern in that patterns do not necessarily containregular and ordered features. Arrays typically, but do not necessarily,comprise at least about 4 to about 10,000,000 features, generally in therange of about 4 to about 1,000,000 features.

The term “fluid” as used herein, as in a “fluid medium,” refers tomatter that is nonsolid and at least partially composed of a liquid. Afluid may contain a solid that is minimally, partially or fullysolvated, dispersed or suspended. Examples of fluids include, withoutlimitation, aqueous liquids (including water per se and salt water) andnonaqueous liquids such as organic solvents and the like. The fluid mayalso be a biological fluid containing cells, biomolecules, or the like.

The term “nucleic acid” refers to a nucleoside, nucleotide, orpolynucleotide, including an oligonucleotide, whether generated innature or synthesized in the laboratory, and as such encompassesnon-natural constructs such as plasmids. The terms are usedinterchangeably herein unless specifically indicated otherwise orcontext dictates a different interpretation. Nucleic acids include thosecontaining 2-deoxy-D-ribose as well as D-ribose, and thus encompasspolydeoxyribonucleotides and polyribonucleotides, respectively, and maycontain any of the conventional purine and pyrimidine bases, i.e.,adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U), aswell as protected forms thereof, e.g., wherein the base is protectedwith a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl,isobutyryl or benzoyl, and purine and pyrimidine analogs, known to thoseskilled in the art and described in the pertinent texts and literature.Common analogs include, but are not limited to, 1-methyladenine,2-methyladenine, N⁶-methyladenine, N⁶-isopentyl-adenine,2-methylthio-N⁶-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluoro-uracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methyl-aminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine. Nucleic acids herein may other typesof modifications as well including, without limitation: modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like; non-natural internucleotide linkages,such as methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, phosphorothioates, phosphorodithioates, aminoalkylphosphoramidates, and aminoalkyl phosphotriesters; functionalizationwith pendant moieties; incorporation of intercalators (e.g., acridine,psoralen, etc.); incorporation of chelators (e.g., metals, radioactivemetals, boron, oxidative metals), and the like.

The term “polypeptide” is intended to include any structure comprised oftwo or more amino acids, and thus includes dipeptides, oligopeptides,and proteins, and these terms are used interchangeably herein unless thetext or context indicates otherwise. The amino acids forming all or apart of a peptide may be any of the twenty conventional, naturallyoccurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid(D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H),isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N),proline (P), glutamine (Q), arginine (R), serine (S), threonine (T),valine (V), tryptophan (W), and tyrosine (Y), as well asnon-conventional amino acids such as isomers and modifications of theconventional amino acids, e.g., D-amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically modified aminoacids, β-amino acids, constructs or structures designed to mimic aminoacids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lacticacid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, and nor-leucine), and other non-conventional aminoacids, as described, for example, in U.S. Pat. No. 5,679,782 toRosenberg et al. Peptides may also contain nonpeptidic backbonelinkages, wherein the naturally occurring amide —CONH— linkage isreplaced at one or more sites within the peptide backbone with anon-conventional linkage such as N-substituted amide, ester, thioamide,retropeptide (—NHCO—), retrothioamide (—NHCS—), sulfonamido (—SO₂NH—),and/or peptoid (N-substituted glycine) linkages. Accordingly, peptidescan include pseudopeptides and peptidomimetics. Peptides can be (a)naturally occurring, (b) produced by chemical synthesis, (c) produced byrecombinant DNA technology, (d) produced by biochemical or enzymaticfragmentation of larger molecules, (e) produced by methods resultingfrom a combination of methods (a) through (d) listed above, or (f)produced by any other means for producing peptides.

The term “substantially” as in, for example, the phrase “substantiallyidentical reservoirs” refers to reservoirs that do not materiallydeviate in acoustic properties. For example, acoustic attenuations of“substantially identical reservoirs” deviate by not more than 10%,preferably not more than 5%, more preferably not more than 1%, and mostpreferably at most 0.1% from each other. Other uses of the term“substantially” involve an analogous definition.

The invention provides a method for transfecting cells using acousticradiation in a manner that enables transfection of a variety of celltypes, including non-mammalian cells and mammalian cells, confluentcells and non-confluent cells. Using sonoporation as described herein,the method enables incorporation of exogenous material into host cells,including, without limitation, plasmids, ribonucleoproteins, and otherspecies. As will be described in detail infra, the method lends itselfto use in high-throughput transfection, in large part because the methodcan be carried out with large numbers of cell-containing reservoirs insuccession.

In one embodiment, the method for transfecting cells comprises: (a)providing a system that includes (i) at least two reservoirs eachcontaining host cells and exogenous material to be introduced into thehost cells via sonoporation-induced transfection, and (ii) an acousticradiation generator to generate and direct acoustic radiation; (b)acoustically coupling the acoustic radiation generator to a first of thereservoirs without simultaneously acoustically coupling the acousticradiation generator to any other of the reservoirs; (c) activating theacoustic radiation generator to generate and direct acoustic radiationinto the first reservoir in a manner that induces sonoporation of thehost cells, thereby facilitating introduction of the exogenous materialinto the sonoporated host cells; (d) acoustically decoupling theacoustic radiation generator from the first reservoir; (e) acousticallycoupling the acoustic radiation generator to a second of the reservoirswithout simultaneously acoustically coupling the acoustic radiationgenerator to any other of the reservoirs; and (f) repeating step (c)with respect to the second reservoir.

Generally, although not necessarily, the first and second reservoirs arecontained within a plurality of reservoirs, and the method is repeatedwith some or all of the reservoirs. When this is the case, the methodincludes an additional step following (f), namely, (g) acousticallydecoupling the acoustic radiation generator from the second reservoir,and repeating steps (b) through (g) with respect to the otherreservoirs. To provide modularity and interchangeability of components,it may sometimes be preferred for the device to be used in conjunctionwith a plurality of removable reservoirs, e.g., tubes in a tube rack orthe like. The reservoirs are arranged in a pattern or an array,typically an array, to provide each reservoir with individual systematicaddressability. While each of the reservoirs may be provided as adiscrete or stand-alone container, in circumstances that require a largenumber of reservoirs, e.g., in a high-throughput transfection method, itis preferred that the reservoirs are contained within an integratedmultiple reservoir unit. The multiple reservoir unit may be a well platewith the individual wells serving as reservoirs. Many well platessuitable for use with the device are commercially available and maycontain, for example, 96, 384, 1536, or 3456 wells per well plate, andhaving a full skirt, half skirt, or no skirt. Well plates or microtiterplates have become commonly used laboratory items. The Society forLaboratory Automation and Screening (SLAS) has established and maintainsstandards for microtiter plates in conjunction with the AmericanNational Standards Institute, including the footprint and dimensionstandards ANSI/SLAS 1-2004. The wells of such well plates are generallyin the form of rectilinear arrays.

The availability of such commercially available well plates does notpreclude the manufacture and use of custom-made well plates in othergeometrical configurations containing at least about 10,000 wells, or asmany as 100,000 to 500,000 wells, or more. Furthermore, the materialused in the construction of reservoirs must be acoustically compatibleas well as compatible with the fluid samples contained therein. Forwater-based fluids, a number of materials are suitable for theconstruction of reservoirs and include, but are not limited to, ceramicssuch as silicon oxide and aluminum oxide, metals such as stainless steeland platinum, and polymers such as polyester, polypropylene, cyclicolefin copolymers (e.g., those available commercially as Zeonex® fromNippon Zeon and Topas® from Ticona), polystyrene, andpolytetrafluoroethylene.

In addition, to reduce the amount of movement and time needed totransfect host cells in each of a plurality of reservoirs in rapidsuccession, it is preferred that the center of each reservoir be locatednot more than about 1 centimeter, e.g., not more than about 1.5millimeters, not more than about 1 millimeter, and not more than about0.5 millimeter from a neighboring reservoir center. These dimensionstend to limit the size of the reservoirs to a maximum volume. Thereservoirs are constructed to contain typically no more than about 1 mL,preferably no more than about 500 μL, and more preferably no more thanabout 250 μL of fluid, and in some cases no more than 100 μL, 50 μL, 25μL, 10 μL, 5 μL, 1 μL, or 0.5 μL of fluid. The volume of fluid medium inthe reservoirs, during operation, is thus in the range of about 0.5 μLto about 500 μL. To facilitate consistency, it is also preferred thatthe reservoirs be substantially acoustically indistinguishable.

An acoustic radiation generator comprising an ultrasonic transducer isused to generate acoustic radiation and direct the acoustic radiationgenerated into the reservoir containing the host cells to betransfected. An ultrasonic transducer typically includes an actuator anda focusing element that concentrates acoustic energy produced by theactuator; examples of actuators include piezoelectric andmagnetorestrictive elements, with piezoelectric transducers generally,although not necessarily, preferred herein. In operation, the actuatoris driven by a signal at an ultrasonic driving frequency and producesultrasonic vibrations in the active physical element. These vibrationsare transmitted into and through an acoustic coupling medium and intothe reservoir housing the fluid sample. A single transducer can be used,or in some cases, multiple element acoustic radiation generatorscomprising transducer assemblies may be used. For example, linearacoustic arrays, curvilinear acoustic arrays or phased acoustic arraysmay be advantageously used to generate acoustic radiation that istransmitted simultaneous to a plurality of reservoirs. In a preferredembodiment, a single acoustic radiation generator is employed. Someexamples of acoustic radiation generators that can be advantageouslyused herein are those incorporated into the Acoustic Droplet Ejection(ADE) systems available from Labcyte Inc. (San Jose, Calif.) anddescribed, for instance, in U.S. Pat. No. 6,416,164 to Stearns et al.;U.S. Pat. No. 6,666,541 to Ellson et al.; U.S. Pat. No. 6,603,118 toEllson et al.; U.S. Pat. No. 6,746,104 to Ellson et al.; U.S. Pat. No.6,802,593 to Ellson et al.; U.S. Pat. No. 6,938,987 to Ellson et al.;U.S. Pat. No. 7,270,986 to Mutz et al.; U.S. Pat. No. 7,405,395 toEllson et al.; and U.S. Pat. No. 7,439,048 to Mutz et al. Examples ofcommercially available ADE systems from Labcyte include the Echo®500-series Liquid Handler systems, including the Echo® 525, the Echo®550, and the Echo® 555 Liquid Handlers.

As explained above, the acoustic radiation generator herein preferablyincludes a focusing element. Any of a variety of focusing means thatinclude curved surfaces or Fresnel lenses known in the art may beemployed in conjunction with the present invention. Such focusing meansare described in U.S. Pat. No. 4,308,547 to Lovelady et al. and U.S.Pat. No. 5,041,849 to Quate et al., as well as in U.S. PatentApplication Publication No. 2002037579.

When transfecting host cells in each of a plurality of reservoirs, as ina well plate or other type of array, the method is carried out inconjunction with a means for positioning each of the reservoirs and anacoustic radiation generator in acoustic coupling relationship, suchthat after each sonoporation event, the acoustic radiation generator isaligned with the next reservoir to be irradiated. The positioning meansmay be incorporated into the transfection system in order to move asubstrate containing the reservoirs (which may be positioned on amovable stage, for instance) relative to the acoustic ejector, or viceversa. Rapid and successive irradiation of reservoirs is thereby readilyfacilitated. Either type of positioning means, i.e., an ejectorpositioning means or a reservoir or reservoir substrate positioningmeans, can be constructed from, for example, motors, levers, pulleys,gears, a combination thereof, or other electromechanical or mechanicalmeans. The reservoir-to-reservoir transition time is preferably at mostabout 0.5 seconds, preferably at most about 0.1 seconds, and optimallyat most about 0.001 seconds.

It should be noted that the acoustic radiation generator must be inacoustic coupling relationship with respect to the reservoir to beirradiated and thus to the reservoir contents as well, and that, whensuccessively irradiating multiple reservoirs, the acoustic radiationgenerator decouples from each irradiated reservoir after sonoporation,and is then acoustically coupled to the next reservoir for the nextsonoporation event. The process thus involves acoustically coupling theacoustic radiation generator to a first reservoir to be irradiated,irradiating the first reservoir, then acoustically decoupling theacoustic radiation generator from the first reservoir, then acousticallycoupling the acoustic radiation generator to the next reservoir,irradiating the next reservoir, etc., and continuing the process untilthe desired number of reservoirs has been irradiated. Although it ispossible to achieve acoustic coupling through direct contact with thecontents of the reservoirs, the preferred approach is to acousticallycouple the acoustic radiation generator to a reservoir and thus to thecontents thereof without allowing any portion of the acoustic radiationgenerator (e.g., the focusing means) to contact the contents of thereservoir.

The acoustic radiation generator may be in either direct contact orindirect contact with the external surface of each reservoir. Withdirect contact, in order to acoustically couple the acoustic radiationgenerator to a reservoir, it is preferred that the direct contact bewholly conformal to ensure efficient acoustic energy transfer. That is,the acoustic radiation generator and the reservoir should havecorresponding surfaces adapted for mating contact. Thus, if acousticcoupling is achieved between the acoustic radiation generator andreservoir through the focusing means, it is desirable for the reservoirto have an outside surface that corresponds to the surface profile ofthe focusing means. Without conformal contact, efficiency and accuracyof acoustic energy transfer may be compromised. In addition, since manyfocusing means have a curved surface, the direct contact approach maynecessitate the use of reservoirs that have a specially formed inversesurface.

Optimally, acoustic coupling is achieved between the acoustic radiationgenerator and each reservoir through indirect contact, as described inU.S. Pat. No. 6,416,164 to Stearns et al.; U.S. Pat. No. 6,666,541 toEllson et al.; U.S. Pat. No. 6,603,118 to Ellson et al.; U.S. Pat. No.6,746,104 to Ellson et al.; U.S. Pat. No. 6,802,593 to Ellson et al.;U.S. Pat. No. 6,938,987 to Ellson et al.; U.S. Pat. No. 7,270,986 toMutz et al.; U.S. Pat. No. 7,405,395 to Ellson et al.; and 7,439,048 toMutz et al., cited supra and incorporated by reference herein.Generally, an acoustic coupling medium is placed between the acousticradiation generator and the base of the reservoir to be irradiated. Theacoustic coupling medium may be an acoustic coupling fluid, preferablyan acoustically homogeneous material in conformal contact with theuppermost surface of the acoustic radiation generator, e.g., with anacoustic focusing means located on the uppermost surface of the acousticradiation generator, and the underside of the reservoir. In addition, itis important to ensure that the acoustic coupling fluid is substantiallyfree of material having different acoustic properties than the fluidmedium in the reservoir being irradiated. In use, a first reservoir isacoustically coupled to the acoustic radiation generator such thatacoustic radiation generated by the acoustic radiation generator isdirected, e.g., by the focusing means, into the acoustic couplingmedium, which then transmits the acoustic radiation into the reservoir.The system may contain a single acoustic ejector, or, as notedpreviously, it may contain multiple ejectors. Single ejector designs aregenerally preferred over multiple ejector designs because accuracy ofdroplet placement and consistency in droplet size and velocity are moreeasily achieved with a single ejector. However, the invention is notlimited to single ejector designs.

As explained earlier herein, a reservoir containing host cells to betransfected via sonoporation contains the host cells as well as theexogenous material to be introduced into the host cells. The host cellsand the exogenous material are advantageously contained in a fluidmedium, i.e., a host cell compatible fluid medium, such as a buffer,e.g., an isotonic buffer such as Dulbecco's phosphate buffered saline(DPBS). By a “compatible” fluid medium is meant one that the cells cansurvive in for at least five minutes.

Host cells and host cell types: Cells should be grown in appropriatemedium with all necessary factors, and the medium must be free ofcontamination. Cell density in the fluid medium contained within areservoir should be optimized, as too low a density can cause poorgrowth in the absence of cell-to-cell contact, and too high a densitycan result in contact inhibition, making cells resistant to uptake ofnucleic acid or other macromolecules. Host cells are commonly derivedfrom cells taken from a subject, such as a cell line. Many types ofmammalian cells can be transfected using the method of the invention,including not only those mammalian cell lines that are commonly workedwith but also, in some cases, cells that are extremely difficult totransfect by prior known methods. Commonly worked with mammalian celllines include, for instance, the human cell lines HeLa, HepG2, HUVEC,MCF7, H1 human embryonic, GM12878, K562, and Jurkat E6.1; the mouse celllines NIH-3T3 and MEFs (mouse embryonal fibroblasts); and other celllines such as Chinese hamster ovary (CHO) cells and African green monkeykidney (COS-7) cells. Cells that are normally very difficult totransfect, but that may be efficiently transfected using the presentmethod, include, by way of example: lymphocytes, including both B-cellsand T-cells; primary cells of all origins; neurons; stem cells of alltypes; and oocytes. Specific cell lines within this latter group includethe human lymphoblastoid lines GM12878 and Jurkat E6.1, and H1 humanembryonic cells.

Specific examples of cell lines that can be transfected using thepresent method, include, without limitation, C8161, CCRF-CEM, MOLT,mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa,MiaPaCell, Pancl, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24,J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1,SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21,DLD2, Raw264.7, NRK, NRK-52E, MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS,COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouseembryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts;10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis,A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7,CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23, COR-L23/CPR,COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82,DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69,HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat,JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48,MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10,NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT celllines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9,SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Verocells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.Cell lines are available from a variety of sources known to those withskill in the art (see, e.g., the American Type Culture Collection (ATCC)(Manassas, Va.).

The exogenous material in the fluid medium containing the host cells,i.e., the exogenous material to be incorporated into the host cells viatransfection, can be any material that can be introduced into a livingcell to provide an intended function, result, or benefit. Whiletransfection is generally defined as introducing molecules into arecipient cell so as to add to, alter, and/or regular the cell's DNA,that definition can be expanded in the present context insofar as themethod of the invention facilitates the incorporation of a wide varietyof molecular moieties into a host cell, including but not limited tomolecular moieties that ultimately affect the structure and function ofhost cell DNA. “Exogenous material,” as that term is used herein, then,includes, without limitation: nucleic acids such as DNA, RNA, mRNA,small interfering RNA (siRNA/RNAi), micro RNA (miRNA), DNA plasmidsencoding genes that will express proteins in the host cell, DNA plasmidsthat serve other purposes like generating enhancers or RNA), smalllinear DNA encoding a moiety of interest such as a homologousrecombination donor for CRISPR; proteins and polypeptides, includingkinases, cytokines, chromatin remodeling enzymes, fluorescent proteinsfor visualization, and mutant versions of normal proteins; and smallmolecules, particularly low molecular weight (<about 900 daltons)organic compounds that are biologically useful (e.g., that may helpregulate a biological process), such as inhibitors or activators ofspecific pathways (e.g., drug or toxin pathways), radioactively labelednucleotides or amino acids, cholesterol, glucose and other sugars, andthe like (see, e.g., the NCBI BioSystems Database entries under “SmallMolecules”); lipidic and saccharidic materials such as lipids,lipoproteins, lipopolysaccharides, lipopolysaccharides, andpolysaccharides; and ribonucleoproteins such as the Cas proteins andprotein complexes used in CRISPR editing.

In a preferred embodiment, the exogenous material comprises a nucleicacid, such as a DNA plasmid, or a ribonucleoprotein, such as a Cas:guideRNA ribonucleoprotein.

Nucleic acids: Exogenous nucleic acids that can be introduced into thehost cells may be in the form of genes, gene fragments, oligonucleotidesand polynucleotides, or antisense oligonucleotides and polynucleotides,or may be any other type of nucleic acid having biological activity orother benefit. The nucleic acids introduced into host cells using thepresent method are generally, although not necessarily, in the form ofconstructs that include at least one structural gene under thetranscriptional and translational control of a suitable regulatoryregion, e.g., a promoter sequence in a vector that may be a plasmid, inturn enabling expression of the peptide or protein encoded by theaforementioned structural gene. Such constructs usually contain one ormore regulatory elements other than promoters, as is known in the art.Most commonly, an exogenous nucleic acid is introduced into a host cellusing the present method by means of a DNA plasmid. Other suitablevectors are known and described in the pertinent texts and literature.Transfection of host cells with a nucleic acid may in some cases requirea transfection facilitator such as a cationic lipid formulation, acationic polymer (e.g., DEAE-dextran or polyethylenimine), a dendrimer,or the like. The method of the invention, as alluded to above, works incombination with CRISPR (clustered regularly interspaced shortpalindromic repeats) plasmids, as established in Example 5. Whentransfecting host cells with CRISPR plasmids, some minor modificationsmay be necessary or desirable; for instance, a CRISPR plasmid, becauseit is relatively large, may be subjected to an upstream treatment toreduce its overall size. In the alternative, or in addition, atransfection helper reagent such as JetPEI® (Polyplus Transfection) maybe used.

CRISPR and RNPs: It should be emphasized that the present methods areuseful in conjunction with a wide variety of proteins, includingribonucleoproteins (RNPs). An RNP is a protein bound to RNA, i.e., it isa complex of a ribonucleic acid and an RNA-binding protein. Suchcomplexes are important in a number of biological functions, includingDNA replication and regulation of gene expression. Of particularsignificance in the present context are engineered RNPs that leveragethe CRISPR-Cas mechanism, which has recently taken on enormoussignificance in the field of genome editing; see, e.g., Donohoue et al.,Trends Biotechnol. (Aug. 1, 2017). As is known in the art, CRISPR-basedtransfection involves the use of RNPs composed of a CRISPR-associatedprotein, or “Cas” protein, and RNA, i.e., a “guide RNA” (gRNA), whichmay either a combination of crRNA (which locates the target sequence ofhost DNA) and tracrRNA (which base pairs with the crRNA to form an RNAduplex), or a single guide RNA (sgRNA), which incorporates both crRNAand tracrRNA.

The Cas protein, usually Cas9 or a homolog thereof, and a guide RNA,either “single guide” RNA (sgRNA) or a combination of crRNA andtracrRNA, are the primary components of a CRISPR transfection system.Variations of the CRISPR components are possible and described, forexample, in U.S. Pat. No. 8,771,945 to Zhang et al., incorporated byreference in its entirety. While Cas9, such as Cas9 from S. pyogenes orS. pneumoniae, is the CRISPR nuclease most commonly used, it will beappreciated that other Cas proteins can be used in place of Cas9,particularly Cfp1, with other Cas proteins including, withoutlimitation, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9(also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2,Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4,Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3,Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modifiedversions thereof. A Cas protein used herein in conjunction with CRISPRtransfection may be altered or modified in one of various ways, e.g., amutated Cas nuclease that lacks the ability to cleave one or bothstrands of a target polynucleotide can be useful in many contexts. Forinstance, CRISPR-Cas9 D10A nickase, containing an aspartate-to-alaninesubstitution (D10A) in the RuvC I catalytic domain of Cas9 from S.pyogenes, cleaves a single strand of a polynucleotide duplex instead ofa double strand. As another example, “dCas9,” which contains theaforementioned mutation as well as an H840A mutation in the HNH domain,completely lacks the ability to cleave a polynucleotide. See, e.g., Qiet al. (2013) Cell 152: 1173-1183. As a further example, the Casnuclease is part of a fusion protein, in which the fused protein domainis selected to provide an added function, e.g., transcription activationor repression activity, nucleic acid binding activity, or the like.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. A guide sequence may beselected to target any target sequence. In some embodiments, the targetsequence is a sequence within a genome of a cell, particularly asequence that is unique in the target genome. The guide sequence may beselected to enable the targeting of a polynucleotide in a host cell forany number of purposes, modifying the target polynucleotide by deleting,inserting, translocating, inactivating, or activating targeted regions.The CRISPR complex therefore has a broad spectrum of applications inmany fields, including gene therapy, drug screening, disease diagnosis,and prognosis. Accordingly, the target polynucleotide of a CRISPRcomplex may include a number of disease-associated genes andpolynucleotides as well as signaling biochemical pathway-associatedgenes and polynucleotides. Many disease-associated genes andpolynucleotides are known in the art, as are signaling biochemicalpathway-associated genes and polynucleotides; see, e.g., U.S. Pat. No.8,771,945 to Zhang et al., supra.

The present method for sonoporating host cells includes a means forimparting the acoustic radiation generated by the acoustic radiationgenerator to the host cells. Generally, although not necessarily, themeans for imparting acoustic radiation to the host cells comprises atransfection excitation material, i.e., a material that is caused tovibrate ultrasonically upon irradiation with the acoustic radiationgenerator, and that transfers the ultrasonic vibrations to neighboringhost cells. The transfection excitation material may comprise aplurality of acoustically activatable moieties in the form of particles,beads, or localized fluid volumes, where a “localized fluid volume”refers to a spatially localized volume of fluid, which may or may not becircumscribed by a delineating feature, and wherein the localized fluidvolume will usually have different physical properties than thesurrounding fluid, although this is not required. Uncircumscribedlocalized fluid volumes include fluidic compositions wherein localizedlipidic, or hydrophobic, regions are contained within a hydrophilic(e.g., aqueous) fluid, or wherein localized hydrophilic (e.g., aqueous)regions are contained within a lipidic, or hydrophobic fluid.Circumscribed localized fluid volumes include fluid-containingmicrocapsules, e.g., liquid-containing and gel-containing microcapsules,wherein the capsule wall may or may not allow for some exchange ofmaterial between the capsule interior and the external fluid, andwherein the fluid may or may not contain suspended particles. Stillother types of circumscribed volumes are comprised of a first fluid thatmay or may not be immiscible with the fluid in which it is contained,wherein a molecular layer of an immiscible material circumscribes thefirst fluid so as to provide a barrier between the fluidic interior andthe fluidic exterior. See, e.g., U.S. Pat. No. 7,270,986 to Mutz et al.

In one embodiment of the invention, the transfection excitation materialcomprises gas-filled microbubbles that are incorporated into the fluidmedium along with the host cells and the exogenous material.

The microbubbles used in a preferred embodiment herein are small spheresencapsulating a gas core within a shell having an outer surface that canbe functionalized, e.g., by attachment of a targeting ligand. Thetargeting ligand will sometimes be hereinafter referred to as a “firstbinding moiety,” insofar as the targeting ligand can associate with asecond binding moiety present on an antibody specific for the host cell,such that association of the first and second binding moieties resultsin a microbubble-host cell complex. Formation of the microbubble-hostcell complex is one technique for ensuring that the acoustic radiationreceived by the microbubbles and causing them to vibrate is transmittedto the host cells, facilitating sonoporation. Without wishing to bebound by theory, transmitting acoustic radiation to the host cells isbelieved to facilitate sonoporation by physically disrupting the cellmembrane or cell wall, creating transient pores that allow cellularuptake of large molecules such as DNA or an RNP.

Examples of typical microbubble materials, i.e., typical shellmaterials, include, without limitation, lipids, polymers, albumin, andgalactose, although lipidic materials are most commonly used. Othertypes of shell materials that are longer lasting, i.e., resistant todegradation (via biodegradation or other processes), can also be used,e.g., coated glass beads or cross-linked polymers (see U.S. Pat. No.5,487,390 to Cohen et al., incorporated by reference herein). Suitablemicrobubbles include microsphere-type products used in medical contrastimaging (i.e., in contrast-enhanced ultrasound), cell isolation, andcell separation. Accordingly, shell materials described in U.S. PatentApplication Publication No. 2015/0219636 A1 to Rychak et al. (applicantTargeson, Inc., San Diego, Calif.), which pertains to use of microbubblecontrast agents in various contexts, can also be used in conjunctionwith the present method. The disclosure of the aforementioned patentapplication is incorporated by reference herein with respect to itsdisclosure of suitable microbubble shell materials and surfacefunctionalization techniques. Preferred microbubble shell materials foruse in conjunction with the invention are relatively elastic in order tominimize the likelihood of cavitation during sonoporation. The gas coreof the microbubble can be a perfluorocarbon, air, or nitrogen, althoughperfluorocarbons are most common. It is the gas core of the microbubblethat oscillates in an ultrasonic frequency field, which in turn causesthe microbubbles to resonate during the present transfection method.Although the underlying mechanism has not been identified with clarity,it should be emphasized that acoustically induced resonance ofmicrobubbles tethered to host cells as described herein is responsiblefor successful transfection.

Commercially available ultrasound contrast agents that can beadvantageously used with the present invention include, by way ofexample: Targesphere® and Targesphere® SA (available from Targeson, SanDiego, Calif.; see Tlaxa et al. (2010) Ultrasound Med. Biol.36(11):1907-18); Optison® (GE Healthcare), albumin microbubbles with anoctafluoropropane gas core; Levovist® (Schering), having alipid/galactose shell and a core of air; Imagent® lipid microsphereswith a perflexane core; Definity® lipid microspheres with anoctafluoropropane gas core; and Lumason® sulfur hexafluoride lipidmicrobubbles (previously Sonovue®) and MicroMarker microbubbles (BraccoImaging S.p.A./Fujifilm Visualsonics). Microbubbles intended for otherpurposes can also be used, such as the streptavidin-coated glassmicrobubbles available from Akadeum Life Sciences (Ann Arbor, Mich.).

In one embodiment of the invention, the microbubbles are conjugated tothe host cells to facilitate transfer of acoustic energy from theirradiated microbubbles to the cells, thereby allowing transfection ofexogenous material into the cells through the excited cell membrane orcell wall. Preparation of microbubble-antibody conjugates typicallyinvolves functionalization of the microbubbles with a first bindingmoiety, followed by combining the functionalized microbubbles withantibodies specific for the host cell type, where the antibodies arefunctionalized with a second binding moiety that links to the firstbinding moiety present on the microbubbles. Mixing is carried out in ahost cell compatible fluid medium. In preparing the microbubble-antibodyconjugates, the mass/volume ratio is typically in the range of about 0.5μg to 5 μg antibody to 2×10⁷ microbubbles, more typically in the rangeof about 0.5 μg to 3 μg antibody to 2×10⁷ microbubbles, and most usuallyin the range of about 0.5 μg to 1.5 μg antibody to 2×10⁷ microbubbles.

Attachment between the first binding moiety and the second bindingmoiety, i.e., the “binding pair” forming the linkage that results in themicrobubble-cell complex, may be covalent or noncovalent, althoughbinding is typically noncovalent. Examples of covalent attachmentinclude an amide linkage formed between a free amino group that servesas one of the first and second binding moieties and a carboxyl groupthat serves as the other of the first and second binding moieties.Noncovalent modes of attachment include, for instance, ionic bonding,hydrogen bonding, adsorption or physical immobilization. Exemplarybinding pairs include any haptenic or antigenic compound in combinationwith a corresponding antibody or binding portion or fragment thereof(e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulin and goatanti-mouse immunoglobulin) and nonimmunological binding pairs (e.g.,biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine andcortisol]-hormone binding protein, receptor-receptor agonist orantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme inhibitor, and complementary polynucleotide pairs capableof forming nucleic acid duplexes), and the like. Biotin-streptavidinattachments are most commonly used, typically using commerciallyavailable biotinylated antibodies, and microbubblessurface-functionalized with streptavidin.

“Loaded” microbubble-antibody conjugates can then prepared by mixing themicrobubble-antibody conjugates with the exogenous material, where theexogenous material is as described earlier herein, e.g., a DNA plasmidor a CRISPR RNP. A preferred concentration/count ratio for loading themicrobubble-antibody conjugates is in the range of about 1-10 μM RNP to1.25×10⁷ conjugates (5×10⁸ per mL), optimally in the range of about 3-6μM RNP to 1.25×10⁷ conjugates. These are representative ranges only, andare not intended to be limiting, insofar as suitable concentration/countratios for any exogenous material can be determined empirically. At thispoint, the concentration of loaded microbubble-antibody conjugates inthe fluid medium can be adjusted, e.g., by dilution with a hostcell-compatible fluid, where the fluid may or may not be the same as thefluid medium used in step (a). The extent of dilution is optimized toprovide an environment conducive to cell health and thus bettertransfection efficiency as well. Optimization of the extent of dilutionis described in the Examples. It should be noted that, in general, asuspension that is too dilute will not provide a sufficient degree oftransfection, while a suspension that is too concentrated will similarlyprovide an insufficient degree of transfection, although for differentreasons; in the latter case, the acoustic energy may not reach many ofthe host cells, as they will essentially be shielded from the toneburstby the microbubble-antibody conjugates.

Sonoporation: The loaded microbubble-antibody conjugates are thenirradiated by activating the acoustic radiation generator to generateand direct acoustic radiation into a reservoir containing the loadedconjugates in a fluid medium as described previously, using sonoporationparameters selected to bring about transfection. Suitable sonoporationparameters can be selected empirically, by correlating observedtransfection efficiency with respect to one or more sonoporationparameters, such as acoustic intensity, transducer output frequency, andtoneburst profile (e.g., toneburst width). For instance, as explainedearlier, the acoustic pressure used in irradiating a reservoircontaining host cells, microbubbles, and the selected exogenousmaterial, should be sufficient to induce resonance of the microbubblesbut, in a preferred embodiment, not be so high as to cause microbubblecavitation in the fluid region in the vicinity of the acoustic focalspot, which is usually located on the inner surface of the reservoirbottom. Typical acoustic pressures at the focal spot are in the range ofabout 1 MPa to about 2 MPa. Optimal sonoporation parameters may bedetermined by those of ordinary skill in the art using routineexperimentation, and will generally vary with cell type. Generally,however, sonoporation is conducted by irradiating the reservoir withshort bursts of a cyclic acoustic toneburst each on the order of tens ofmilliseconds or less and occurring at about 10 to about 25 times persecond for about 15-40 seconds, e.g., irradiating at less than 1 msduration 10 times per second (10 Hz) for about 30 seconds. By way ofillustration, Example 6 describes sonoporation with using theaforementioned protocol, irradiating with 300 cyclic acoustic toneburstsat a burst repetition rate of 10 Hz (corresponding to a sonoporationtime period of about 30 seconds, as indicated above), with eachtoneburst consisting of 8 cycles of output. In Example 6, the toneburstduration was approximately 3.5 μs (8 cycles divided by a nominal outputfrequency of 2.25 MHz). Irradiation can be repeated within any onereservoir, changing the location of the focal point or the width of thebeam, if desired, to maximize the number and area of microbubbles thatare sonoporated, in turn maximizing transfection efficiency.

Usually, it is preferred that the acoustic radiation generated be of afrequency and intensity selected to ensure that irradiated microbubblesreceive excitation radiation having a wavelength within about 15% of theaverage resonance frequency of the microbubbles in the reservoir,preferably within about 5% of the average resonance frequency of themicrobubbles in the reservoir, or within about 15% of a harmonic of theaverage frequency of the microbubbles in the reservoir, preferablywithin about 5% of a harmonic of the average frequency of themicrobubbles in the reservoir. In a related embodiment, the acousticradiation generated may be of a frequency and intensity selected toensure that irradiated microbubbles of a particular size or within aparticular size range receive excitation radiation having a wavelengthwithin about 15% of the average resonance frequency of the microbubblesin the reservoir, preferably within about 5% of the average resonancefrequency of the microbubbles in the reservoir, or within about 15% of aharmonic of the average frequency of the microbubbles in the reservoir,preferably within about 5% of a harmonic of the average frequency of themicrobubbles in the reservoir. If the microbubbles are in a compositionwith a multimodal size distribution, they can be irradiated more thanonce with each irradiation event targeting microbubbles having or neareach modal peak.

In addition, while some microbubbles may undergo cavitation as a resultof irradiation, it is generally preferred that cavitation be avoided. Assuch, sonoporation is usually conducted by adjusting the acousticradiation generator to irradiate the microbubbles using an acousticsonoporation pressure in the range of about 50% to 90% of the minimumacoustic pressure that would result in microbubble cavitation. Theacoustic sonoporation pressure may be, for example, in the range ofabout 0.2 MPa to about 2 MPa, typically less than about 1.5 MPa. Whilefor many cell lines acoustic power levels below the cavitation limitwill provide good transfection results via sonoporation, an additionalbenefit of operating below the cavitation limit is that re-use of themicrobubbles is then possible. That is, when irradiating withsubcavitation acoustic energy, the number of intact microbubblesremaining after irradiation is generally within about 50%, 80%, 90%, or99% of the original number of microbubbles prior to sonoporation.Post-sonoporation intact microbubbles can be re-used with the same hostcells, which may or may not be in the same reservoir as the initiallysonoporated host cells. Alternatively, if post-sonoporation intactmicrobubbles are separated from the conjugating antibody, eithernaturally or as a result of treatment, they can be re-used with adifferent host cell type. Operating at subcavitation acoustic levelsalso allows for repetition of irradiation in the same reservoir,wherein, for instance, after an initial sonoporation event, an acousticbeam generated by the acoustic radiation generator is used to bringintact, already irradiated microbubbles into contact with host cells ina different spatial location within the reservoir.

In other embodiments of the invention, as will be explained infra,sonoporation of does not require repetition of acoustic coupling anddecoupling steps. The above description regarding host cells, exogenousmaterial, fluid medium, transfection excitation material, and otherelements and aspects of the transfection methodology is otherwiseapplicable to the following embodiments:

In an additional embodiment of the invention, then, an acoustic methodis provided for transfecting cells by: acoustically coupling an acousticradiation generator to a reservoir that contains host cells, exogenousmaterial to be transfected into the host cells, and a fluid medium; andactivating the acoustic radiation generator to generate acousticradiation and direct the acoustic radiation into the reservoir in amanner that induces sonoporation of the host cells without resulting ina temperature increase in the fluid medium of greater than about 10° C.For instance, the method can induce sonoporation without resulting in atemperature increase of greater than about 5° C., 2° C., or 1° C. In arelated embodiment, sonoporation takes place without raising thetemperature of the fluid medium to greater than about 40° C. It ispreferred that the acoustic radiation generated is directed into thereservoir using a focusing means, such that sonoporation is carried outusing focused acoustic radiation. It is also preferred that the fluidmedium contain a transfection excitation material as explained earlierherein.

In another embodiment, an acoustic method is provided for transfectingcells by acoustically coupling an acoustic radiation generator to aselected reservoir contained within an integral multiple reservoir unitcomprising at least 1536 reservoirs, the selected reservoir containinghost cells, exogenous material to be transfected into the host cells,and a fluid medium; and activating the acoustic radiation generator togenerate acoustic radiation and direct the acoustic radiation into thereservoir in a manner that induces sonoporation of the host cells,thereby facilitating incorporation of the exogenous material into thesonoporated host cells. The integral multiple reservoir unit may,accordingly, be a microwell plate with 1536 wells, or with 3456 wells,or the like. As in the preceding embodiment, it is preferred that theacoustic radiation generated is directed into the reservoir using afocusing means and that the fluid medium contain a transfectionexcitation material.

In another embodiment, an acoustic method is provided for transfectingcells by acoustically coupling an acoustic radiation generator to areservoir that contains host cells, exogenous material to be transfectedinto the host cells, a fluid medium, and a transfection excitationmaterial comprised of a plurality of acoustically activatable localizedfluid volumes; and activating the acoustic radiation generator togenerate acoustic radiation and direct the acoustic radiation into thereservoir in a manner that acoustically activates the localized fluidvolumes so that they vibrate at a frequency that is within about 15% ofthe average resonance frequency of the localized fluid volumes or withinabout 15% of a harmonic of the average resonance frequency of thelocalized fluid volumes, thereby facilitating incorporation of theexogenous material into host cells in the proximity of the acousticallyactivated localized fluid volumes. For instance, the localized fluidvolumes may be acoustically activated so that they vibrate at afrequency that is within about 5% of the average resonance frequency ofthe localized fluid volumes or within about 5% of a harmonic of theaverage resonance frequency of the localized fluid volumes. Again, in apreferred embodiment, the acoustic radiation directed into the reservoiris focused acoustic radiation.

In another embodiment, an acoustic method for transfecting cells isprovided that comprises: acoustically coupling an acoustic radiationgenerator to a reservoir that contains host cells, exogenous material tobe transfected into the host cells, a fluid medium, and a transfectionexcitation material comprised of a plurality of acoustically activatablelocalized fluid volumes having a size distribution; and activating theacoustic radiation generator to generate acoustic radiation and directthe acoustic radiation into the reservoir in a manner that acousticallyactivates the localized fluid volumes having a size within about 15% ofa selected size, thereby facilitating incorporation of the exogenousmaterial into host cells in the proximity of the acoustically activatedlocalized fluid volumes.

In a related embodiment, an acoustic method for transfecting cells isprovided that comprises: (a) acoustically coupling an acoustic radiationgenerator to a reservoir that contains host cells, exogenous material tobe transfected into the host cells, a fluid medium, and a transfectionexcitation material comprised of a plurality of acoustically activatablelocalized fluid volumes having a multimodal size distribution; (b)activating the acoustic radiation generator to generate acousticradiation and direct the acoustic radiation into the reservoir in amanner that acoustically activates localized fluid volumes having a sizethat is within about 15% of a first modal peak, whereby the acousticallyactivated localized fluid volumes transfer acoustic energy to nearbyhost cells; (c) repeating step (b) to acoustically activate localizedfluid volumes having a size that is within about 15% of a second modalpeak; and (d) optionally repeating step (b) to acoustically activatelocalized fluid volumes having a size that is within about 15% of one ormore additional modal peaks.

In an additional embodiment, an acoustic method is provided fortransfecting cells, the method comprising: acoustically coupling anacoustic radiation generator to a reservoir containing host cells,exogenous material to be transfected into the host cells, a fluidmedium, and a transfection excitation material comprised of a pluralityof acoustically activatable localized fluid volumes having a sizedistribution; and activating the acoustic radiation generator togenerate acoustic radiation having a selected frequency content anddirect the acoustic radiation generated into the reservoir in a mannerthat induces sonoporation of the host cells, wherein the frequencycontent of the acoustic radiation generated is selected to correlatewith the size distribution of the acoustically activatable localizedfluid volumes. By “correlate with” is meant that individual frequencieswithin the acoustic radiation are tuned to target and acousticallyactivate individual sizes and size ranges within the localized volumedistribution.

In a related embodiment, an acoustic method for transfecting cells isprovided by acoustically coupling an acoustic radiation generator to areservoir containing host cells, exogenous material to be transfectedinto the host cells, a fluid medium, and a transfection excitationmaterial comprised of a plurality of acoustically activatable localizedfluid volumes having a spatial distribution within the reservoir; andactivating the acoustic radiation generator to generate acousticradiation having a selected frequency content and direct the acousticradiation generated into the reservoir in a manner that inducessonoporation of the host cells, thereby facilitating incorporation ofthe exogenous material into the sonoporated host cells, wherein thefrequency content of the acoustic radiation generated is selected tocorrelate with the spatial distribution of the acoustically activatablelocalized fluid volumes. In this case, “correlate with” indicates thatindividual frequencies within the acoustic radiation are tuned to targetand acoustically activate localized volumes at different locationswithin the reservoir.

In another embodiment, sonoporation is conducted using two transducersoperating in concert (preferably but not necessarily simultaneously) butat different frequencies, wherein one of the transducers is an annulartransducer is operably mounted around and enclosing a standardtransducer. In this embodiment, the annular transducer and the standardtransducer will generally operate at different frequencies. Forinstance, when the sonoporated cells are to be acoustically ejected fromthe fluid medium, the annular transducer may operate at a frequencyselected to bring about sonoporation, while the standard transducer canbe operated at a frequency effective to result in acoustic ejection ofsonoporated cells, e.g., into a reservoir, onto a substrate, or fortransport to an analytical instrument for analysis. In such a case, theannular transducer may operate at a frequency in the range of about 1MHz to about 2.5 MHz, and the standard transducer may operate at afrequency in the range of about 6 MHz to about 20 MHz, preferably in therange of about 9 MHz to about 14 MHz, and optimally about 11.5 MHz.

In one aspect of this embodiment, one of the two transducers primarilyfunctions to supply the acoustic energy for sonoporation and the othertransducer delivers acoustic energy to change the relative position ofthe microbubbles with respect to the host cells when microbubble-cellconjugation is not used. Ideally, when the microbubbles and the hostcells are positioned in proximity of each other, it should be in aregion of acoustic intensity effective to cause sonoporation.

In an additional embodiment, sonoporation involves irradiation withmultiple acoustic tonebursts in succession, each having a differentacoustic frequency effective to sonoporate differently sizedmicrobubbles. The acoustic frequency of each of the multiple acoustictonebursts is typically in the range of about 1.5 MHz to about 5.0 MHz,more usually in the range of about 2.0 MHz to about 2.5 MHz. A narrowdistribution of microbubble sizes typically requires a smaller range ofacoustic frequencies to achieve the same level of excitation as a broaddistribution of microbubble sizes, where acoustic frequency would needto be varied to achieve the same effect. Optimally, the acousticfrequency content is adjusted in response to the distribution ofresonance frequencies for the microbubbles to improve the uniformity ofsonoporation and at the minimal amount of total delivered acousticenergy. The multiple acoustic tonebursts are commonly 5-cycle to10-cycle tonebursts, and may be the same or different.

In a further embodiment, an acoustic method for transfecting cells isprovided that comprises: acoustically coupling an acoustic radiationgenerator to a selected reservoir containing host cells, exogenousmaterial to be transfected into the host cells, a fluid medium, and atransfection excitation material comprising a plurality of acousticallyactivatable localized fluid volumes; and activating the acousticradiation generator to generate acoustic radiation and direct theacoustic radiation into the reservoir in a manner that acousticallyactivates the localized fluid volumes, thereby facilitatingincorporation of the exogenous material into host cells in the proximityof the acoustically activated localized fluid volumes, wherein theacoustic radiation generated is at an acoustic sonoporation pressureselected to ensure that at least 50% of the localized fluid volumesremain intact after irradiation. In one aspect of this embodiment, theacoustic sonoporation is in the range of about 50% to about 90% of theminimum acoustic pressure that would result in cavitation of thelocalized fluid volumes.

The present disclosure is also intended to encompass various ways ofoptimizing the acoustic transfection process. For example, as describedin U.S. Pat. No. 6,932,097 to Ellson et al., U.S. Pat. No. 6,938,995 toEllson et al., U.S. Pat. No. 7,354,141 to Ellson et al., U.S. Pat. No.7,899,645 to Qureshi et al., U.S. Pat. No. 7,900,505 to Ellson et al.,U.S. Pat. No. 8,107,319 to Stearns et al., U.S. Pat. No. 8,453,507 toEllson et al., and U.S. Pat. No. 8,503,266 to Stearns et al., anacoustic radiation generator as described herein can be utilized forcharacterization of a fluid in a reservoir, to measure the height of thefluid meniscus as well as other properties, such as fluid volume,viscosity, density, surface tension, composition, acoustic impedance,acoustic attenuation, speed of sound in the fluid, etc., any or all ofwhich can then be used to determine optimum sonoporation parameters,including acoustic power, acoustic frequency, toneburst duration, and/orthe F-number of the focusing lens. As another example, U.S. Pat. Nos.7,717,544 and 8,770,691 to Stearns et al. describe a method foroptimizing the amplitude of acoustic radiation used for acoustic dropletejection or other acoustic processes, by analyzing the waveforms ofacoustic radiation reflected from surfaces within the reservoir. Inaddition, U.S. Pat. No. 7,481,511 to Mutz et al. and U.S. Pat. No.7,784,331 to Ellson et al. provide methods for controlling acousticprocess parameters to account for variations in reservoir properties.

It is to be understood that while the invention has been described inconjunction with a number of specific embodiments, the foregoingdescription as well as the examples that follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications will be apparent to those skilled in theart. All patents, patent applications, and publications mentioned hereare hereby incorporated by reference in their entireties.

EXPERIMENTAL

Materials:

The following list indicates the materials used in these examples, alongwith the material sources:

Targesphere® SA: Cationic dispersion of streptavidin-functionalizedmicrobubbles (Targeson)

Anti-CD51: Biotinylated anti-human CD51 antibody, 0.5 μg/μL in DPBScontaining 0.09% sodium azide (Biolegend #327906)

HEK-293 cells: Human embryonic kidney cells, cell line 293, in astandard cell culture buffer, DMEM with high glucose media, supplementedwith 4 mM L-Glutamine, 10% fetal bovine serum (FBS), and 100 U/μLpenicillin-streptomycin (ATCC #CRL-1773)

DPBS: Dulbecco's phosphate-buffered saline, with calcium and magnesium(ThermoFisher Scientific #14040182)

GFP: green fluorescent protein (eGFP, or enhanced GFP, was usedthroughout)

gWiz-GFP: eGFP-coded plasmid (Aldevron)

CRISPR plasmid: pCas-Guide-EF1a-eGFP (Origene #GE100018)

Lipofectamine® 3000 (ThermoFisher Scientific)

General Protocol for Examples 1-5:

(1) Preparation of microbubble/DPBS dispersion: A vial containing theTargesphere SA microbubble dispersion was gently shaken and invertedend-to-end for 10 seconds, until the mixture appeared uniformly opaque.100 μL of the Targesphere SA dispersion was extracted and introducedinto a new 1.5 mL tube. 900 μL DPBS was added, and the dispersion gentlymixed. The mixture was then incubated upright at room temperature for 2minutes, followed by spinning in a Minifuge® for 2 minutes to separatethe microbubbles from the suspension liquid. Using a syringe needle, theinfranatant below the white cake of microbubbles was slowly removed, andthe microbubbles were then resuspended in 100 μL of DPBS. (The originalmedium was removed from the Targesphere SA microbubble dispersion andreplaced with DPBS, in order to reduce surface tension problems andadherence of the bubbles to well surfaces seen with the originalmicrobubble medium.) A modified version of this protocol was used inExample 6, as explained in that example.

(2) Conjugation of microbubbles to biotinylated antibody: 10 μganti-CD51 biotinylated antibody (i.e., 20 μL of the 0.5 μg/μL solution)were added to 100 μL of the microbubble dispersion, such that themass/vol ratio of anti-CD51 antibody to the Targesphere SA dispersionwas 1:10. The vial was incubated at room temperature with gentleagitation for about 20 minutes. A modified version of this protocol wasused in Example 6, as explained therein.

(3) Preparation of plasmid-loaded microbubbles (Examples 1-5): The vialcontaining the biotinylated antibody/Targesphere composition wasinverted several times to uniformly mix, and 24 μL of gWiz-GFP plasmidat 5 μg/μL (corresponding to 120 μg gWiz-GFP) was added to the 120 μL ofmicrobubble-biotinylated antibody conjugates prepared in (2), such thatthe mass/vol ratio of plasmid to the Targesphere SA dispersion was 1:1.The vial was again incubated at room temperature with gentle agitationfor about 25 minutes.

(4) Incubation with cells: The following plasmid-loaded microbubbledilutions were prepared:

1:4 vol/vol, 100 μL microbubble dispersion with 300 μL DPBS;

1:20 vol/vol, 20 μL microbubble dispersion with 380 μL DPBS;

1:40 vol/vol, 10 μL microbubble dispersion with 390 μL DPBS; and

1:200 vol/vol, 2 μL microbubble dispersion with 398 μL DPBS.

400 μL DPBS, without microbubbles, was used as a control.

20 μL of the plasmid-loaded microbubbles (or control) were pipetted ontothe plated HEK-293 cells, which had been cultured to 80% confluence in a384-well plate (giving approximately 25,000 cells per well, with amaximum volume of approximately 115 μL/well), media having been removedfrom the cells via pipetting first. In the following table, treatmentconcentration is correlated with microbubble dilution, loading volumeper well, and microbubble-to-cell incubation ratio:

TABLE 1 Treatment Conc. (plasmid-loaded Microbubble Loading Vol.Microbubble:Cell microbubble/mL) Dilution per Well (μL) Incubation Ratio1 × 10⁷  1:200 20 10 5 × 10⁷ 1:40 20 50 1 × 10⁸ 1:20 20 100 5 × 10⁸ 1:4 20 500

The well plate was flipped upside down to facilitate binding, and wasplaced in a 37° C. incubator for 5 minutes. Originally, the plate wasthen washed with 50 μL DPBS to remove unbound microbubbles; having foundthat the washing step also removed cells, however, the washing step wasdiscontinued (note: this may or may not be true for different celltypes).

(5) Sonoporation: The wells were refilled with 80 μL pre-warmed DPBS fora total of 100 μL, and spun at 125 RCF for 5 minutes to remove largebubbles (higher spin speeds would likely have killed many if not most ofthe cells). The cells were then pulsed with ultrasound using an acousticradiation generator in a modified version of an acoustic liquid handler(Echo® 500 series liquid handler, Labcyte Inc., San Jose Calif.), andthe well plates returned to the incubator overnight. After 1-2 days,cells were examined for GFP fluorescence, indicative of DNA uptake, andsurvival.

Example 1

Using the above protocol, four plates of HEK-293 cells were tested. Allfour plates were set up identically, including DPBS-only control wells.The control plate was not sonicated. 384-well plates were used, with25,000 HEK-293 cells per well. Each plate was pulsed with a singlevoltage, either 0 V (control plate), 0.5 V (low power), 1.0 V (mediumpower), or 1.5 V (high power), where 1.0 V resulted in an acousticpressure at the focal spot of about 1.5 MPa. 24 hours post-sonoporation,the cells were examined for GFP fluorescence. It was found thatsonoporation was successful in enabling the HEK-293 cells to take up andexpress the GFP plasmid, and that the percent uptake increased withvoltage and the concentration of plasmid-loaded microbubbles. Thehighest degree of fluorescence was found with the highest concentrationof plasmid-loaded microbubbles and at the highest voltage. Green(fluorescent) cells were concentrated around the perimeter of the wellsdue to the distribution of the microbubbles following inversion of thewell plate. Some cells in the control plate turned green, but very few;this was due to a small percentage of cells taking up plasmid in themedia.

Example 2

The procedures of the General Protocol and Example 1 were follows,except that a “plasmid only” control was substituted for the 1:200dilution, i.e., plasmid was added at the same concentration found in the1:4 wells. The results obtained confirmed that high media concentrationsof plasmid alone were not sufficient to transform cells.

Example 3

The procedures of the General Protocol Example 1 were repeated, butrather than single pulses applied to the center of each well, each wellwas pulsed several times at different locations. This was found toeliminate the concentration of transfected cells around the perimeter ofthe well, providing for a more even distribution, as could be inferredfrom the presence of GFP-fluorescence throughout each well.

Example 4

The procedure of Example 1 was repeated, with the followingmodifications:

Two positive control cell populations were included: (1) cells that weretransfected using Lipofectamine 3000 and following the transfectionprotocol provided by the manufacturer; and (2) dead cells killed by heatshock.

In addition, 1-2 days after sonoporation, cells were stained with a cellmembrane integrity dye that positively stains dead cells, i.e., MultiCytCell Membrane Integrity Dye Panel FL3 dye (IntelliCyt, Albuquerque, N.Mex.).

Transfection and survival rates were analyzed usingfluorescence-activated cell sorting (FACS); results are provided inFIGS. 1-7. FIG. 1 shows a plot of forward versus side scatter height,enabling differentiation of the HEK-293 cells from microbubbles andother material; dead cells appear as a distinct grouping higher on theSSC-H axis, above the denser cluster of live cells. In FIG. 2, the cellsthat were transfected with the GFP-encoding plasmid were detected in theFL1 channel, while cells stained with cell membrane integrity dye, i.e.,dead cells, were detected in the FL3 channel. The “GFP+” and “dead”labels on the plot were set up manually; these control cell populationsare separately illustrated in FIG. 3. As may be concluded from FIG. 4and FIG. 5, the fraction of host cells that were successfullytransfected increased with both acoustic power and microbubbleconcentration. Also, as indicated in FIG. 6, the percentage of livecells remaining post-transfection is near 100%, even at the highestvoltage used, 1.5 V. FIG. 7 illustrates the data obtained for thenegative control, i.e., DPBS only, in the absence of microbubbles.

Example 5

This example describes transfection of a CRISPR plasmid usingsonoporation. The procedures of the General Protocol and Example 1 werefollowed, except that a CRISPR plasmid expressing Cas9 and GFP wassubstituted for the gWiz-GFP plasmid. A 1.5 V sonoporation pulse wasused. A dispersion of microbubble-antibody conjugates was prepared inDPBS (as described in the General Protocol), with a concentration of5×10⁸ microbubble-antibody conjugates per mL of DPBS. This dispersionwas then combined with plasmid at the following ratios, given in μgplasmid per μL of dispersion: 1:1; 2:1; 4:1; and 8:1. Three replicateswere performed at each ratio. One “no plasmid” negative control row andone “no sonoporation” negative control column were included. As inExample 4, results were assayed using IntelliCyt FACS. GFP positivecells were indicative of CRISPR plasmid uptake, and fluorescent dyestain indicated dead cells.

The results are shown in FIGS. 8 and 9. FIG. 8 shows the percentage ofGFP-positive cells obtained for each of the four plasmid concentrations,and FIG. 9 shows the average percentage of GFP-positive cells at eachconcentration, with standard deviation error bars indicated. Thepercentage of GFP-positive cells is above background, indicating thatthe cells have taken up and are expressing the CRISPR plasmid. The dataobtained is also summarized in Table 2:

TABLE 2 Microbubble-to- cell incubation Repli- Repli- Repli- No ratiocate 1 cate 2 cate 3 Pulse 0 0 0 0 0   0.5X 5 7 2 0 1X 5 21 9 3 2X 4 6 710 4X 16 7 24 6

The singlet cell count data was as follows in Table 3:

TABLE 3 Microbubble-to- cell incubation Repli- Repli- Repli- No ratiocate 1 cate 2 cate 3 Pulse 0 2363 4658 3407 4185   0.5X 3082 5199 36555770 1X 3640 3664 6106 6129 2X 3250 5022 6247 6175 4X 2451 2479 35236843

The cell death rate after treatment remains low. FIG. 10 shows thepercentage of dead cells for each of the four plasmid concentrations.The data obtained is also summarized in Table 4:

TABLE 4 Microbubble-to- cell incubation Repli- Repli- Repli- No ratiocate 1 cate 2 cate 3 Pulse   0.5X 3.51% 0.41% 0.44% 0.36% 1X 0.39% 0.00%0.03% 0.00% 2X 0.33% 0.00% 0.00% 0.02% 4X 0.31% 0.02% 0.05% 0.02%

The trend in GFP-positive cell percentage increases with plasmidconcentration and is above the background negative control, confirmingthat CRISPR plasmids can be transfected using sonoporation. An increasein transfection success rate should be possible by using a method thatis not plasmid-based, such as, for example, a technique involving theuse of sonoporation to introduce ribonucleoproteins into target cells.

Example 6

This example describes transfection of HEK-293 cells with a CRISPRCas9/guide RNA ribonucleotide (RNP), using the Alt-RTMS.p. Cas9 Nuclease3NLS obtained from Integrated Technologies, Inc. (IDT, Coralsville,Iowa), and the Alt-R™ CRISPR-Cas9 kit (also obtained from IDT), whichincludes the Alt-R™ CRISPR-Cas9 HPRT positive control crRNA targetingthe HPRT gene, the Alt-R™ CRISPR-Cas9 negative control crRNA, andnuclease-free buffer. A fluorescently labeled tracrRNA for complexingwith the crRNA was obtained separately (Alt-R™ CRISPR-Cas9 tracrRNAconjugated to ATTO™550, also from IDT).

(a) Preparation of microbubble/DPBS dispersion: This is a modifiedversion of the method described in the General Protocol for preparationof the microbubble/DPBS dispersion. A vial containing the Targesphere SAmicrobubble dispersion (concentration 2×10⁹ microbubbles/mL) was gentlyshaken and inverted end-to-end for 10 seconds, until the mixtureappeared uniformly opaque. 50 μL of the Targesphere SA dispersion wasextracted and introduced into a new 1.5 mL tube. 950 μL DPBS was added,and the dispersion gently mixed. The mixture was then incubated uprightat room temperature for 2 minutes, followed by spinning in a Minifuge®for 2 minutes to separate the microbubbles from the dispersion liquid.Using a syringe needle, the infranatant below the white cake ofmicrobubbles was slowly removed until the volume reached 50 μL.

(b) Conjugation of microbubbles to biotinylated antibody: This is amodified version of the method described in the General Protocol forconjugation of microbubbles to biotinylated antibody. 5 μg anti-CD51biotinylated antibody (i.e., 10 μL of a 0.5 μg/μL solution) were addedto 50 μL of the microbubble/DPBS dispersion, such that the mass/countratio of anti-CD51 antibody to the Targesphere SA microbubbles was1:2×10⁷. The vial was incubated at room temperature with gentleagitation for about 20 minutes.

(c) Formation of guide RNA: The crRNA and tracrRNA were suspended in theIDT nuclease-free duplex buffer at 200 μM. The crRNA were combined in a1:1 equimolar ratio, for a final guide RNA concentration of 100 μM:

TABLE 5 HPRT Non-targeting (NT) gRNA, for 10 gRNA, for 5 Componentreactions reactions HPRT crRNA (200 μM) 7 μL — NT crRNA (200 μM) — 4 μLtracrRNA-ATTO 550 (200 μM) 7 μL 4 μL 14 μL  8 μL

The mixture was heated at 95° C. for five minutes, and then removed fromheat and allowed to cool to room temperature.

(d) Formation of the RNP complex: The Cas9 was combined with the guideRNA to create RNPs as described in the Alt-R™ CRISPR-Cas9 User Guide.Briefly, for each well undergoing sonoporation, the guide RNA (i.e., thecrRNA:tracrRNA duplex prepared in the preceding step) were diluted inDPBS, with the Cas9 added last and slowly, at a Cas9: gRNA molar ratioof about 1:1.15. The mixture was then incubated at room temperature for20 minutes. Concentrations were as follows:

TABLE 6 Vol. required HPRT gRNA, NT gRNA, per for 10 for 5 Componentreaction reactions reactions DPBS 2.1 μL 21 μL 10.5 μL  Conjugated 1.2μL 12 μL 6.0 μL crRNA:tracrRNA (100 μM) Cas9 nuclease 1.7 μL 17 μL 8.5μL (61 μM) 5.0 μL 50 μL  25 μL

(e) Preparation of loaded microbubbles: The vial containing theantibody-microsphere conjugates (“aCD51 Targespheres”) was gentlyflicked to mix. The antibody-microsphere conjugates were then combinedwith the RNP prepared in the preceding step, incubated for 20 minutes,and then diluted with DPBS to a final concentration of 4 μM RNP and5×10⁸ microbubble (mb)/mL:

TABLE 7 HPRT HPRT gRNA, no NT gRNA, microbubbles, gRNA, for 4 for 4 for4 Component reactions reactions reactions aCD51 Targespheres 25 μL — 25μL RNPs (20 μM) 20 μL 20 μL 20 μL Incubate 20 min. DPBS with Ca/Mg 55 μL80 μL 55 μL Total volume 100 μL  100 μL  100 μL 

(f) Incubation of the aCD51 Targespheres with HEK-293 cells: Growthmedia (see the General Protocol) was removed from the HEK-293 cells bypipetting. The aCD51Targespheres (i.e., the antibody-conjugatedmicrobubbles) were pipetted up and down to mix well. 25 μLaCD51Targespheres were then pipetted into each well of a 384-wellpolypropylene microwell plate coated with a tissue-culture coating. Theplate was flipped upside down to facilitate binding of the aCD51Targespheres to the cells, and placed in a 37° C. incubator for fiveminutes.

(g) Sonoporation: Using reverse pipetting, 75 μL of pre-warmed completegrowth media was added to each well, bringing the total fluid volume perwell to 100 μL, with an RNP concentration of 1 μM. Sonoporation wascarried out using a 2.25 MHz transducer, a 0.5″ aperture diameter, and aone-inch focal length (F-number of 2). The transducer was activated toirradiate each reservoir with 300 tonebursts at a burst repetition rateof about 10 Hz, meaning that the tonebursts were spaced apart by 0.1sec. Each toneburst consisted of 8 cycles of output, corresponding to atoneburst duration of approximately (8/2.25) 3.5 μs. RF frequency wasconstant throughout the burst output.

(h) Analysis: The rate of successful CRISPR editing was assessed bymeasuring the number of indels introduced into the HPRT gene, where an“indel” is a change in DNA sequence caused by the insertion or deletionof nucleotides. This was quantified using a T7 endonuclease I (T7E1)digestion assay, which cleaves double-stranded DNA if the two halves ofthe helix do not perfectly base pair with each other (i.e. if one halfcontains an indel). After digestion with T7E1, the DNA either remainsintact or is cleaved into two smaller fragments. The digested DNA isanalyzed using gel electrophoresis to separate the fragments by size,and the amount of cleaved versus full length fragments was analyzed.

T7 endonuclease I CRISPR indel detection assay: Cells were washed withDPBS and lysed using Epicentre QuickExtract DNA extraction solution. Theresulting genomic DNA was PCR amplified using primers flanking the siteof interest (i.e., the HPRT gene locus). The PCR products were heated to95° C. to denature them and then slowly cooled to room temperature,encouraging the formation of mismatched pairs. T7 endonuclease I wasthen added and incubated at 37° C. for 1 hour, cleaving anydouble-stranded DNA with a >1 base pair mismatch. The digested DNA wasthen diluted and run out on the AATI Fragment Analyzer using the CRISPRDiscovery Gel Kit to measure percent cleavage.

To measure percent cleavage, the AATI ProSize software compared theamount of “full length” DNA present versus that found in smaller“fragment 1” and “fragment 2” bands. This cleavage percentage is a proxyfor the percent editing performed by CRISPR in the cell population.However, the T7E1 enzyme cannot detect 1 base pair indels, which cancomprise up to 30% of CRISPR editing events. Thus, the cleavagepercentage as measured by this assay is a significant underestimation ofthe true rate of indel formation.

${Cleavage}\mspace{14mu} {\% = \frac{{{{mean}\mspace{14mu} {nmol}\mspace{14mu} {in}\mspace{14mu} {Fragment}\mspace{14mu} 1}\&}\mspace{11mu} 2}{{{nmol}\mspace{14mu} {in}\mspace{14mu} {Full}\mspace{20mu} {Length}} + \left( {{{{mean}\mspace{14mu} {nmol}\mspace{14mu} {in}\mspace{14mu} {Fragment}\mspace{20mu} 1}\&}\mspace{11mu} 2} \right)}}$

The experimental work was repeated and a second set of resultsgenerated. The two experiments are referred to below as Run 1 and Run 2.

Results: FIG. 11 shows a capillary electrophoresis gel illustrating theresults of the mismatch cleavage assay and analysis for Run 1; FIG. 12is a capillary electrophoresis gel providing the results for Run 2. The1,083 bp fragment is the full length PCR product, while “fragment 1” is827 bp and “fragment 2” is 256 bp. Both figures show cleavage in theexperimental samples and a lack of cleavage in all controls, indicatingthat CRISPR transfection and editing was successful. Table 8 gives thepercent cleavage for each run:

TABLE 8 Run 1 Run 2 Experiment: HPRT rep1 27.50% 20.18% HPRT gRNA HPRTrep2 20.37% 27.81% HPRT rep3 17.33% 24.56% No microbubbles HPRT no mb,rep1 4.69% 6.21% HPRT no mb, rep2 8.61% 6.45% HPRT no mb, rep3 5.26%6.36% Non-targeting gRNA NT rep1 5.05% 3.98% NT rep2 4.51% 4.61% NT rep34.47% 2.56% No volts HPRT 0 V 4.99% 8.04% HPRT no mb, 0 V 4.79% 7.92% NT0 V 4.81% 3.79% No treatment No treatment 4.27% 2.71% No treatment 4.09%5.70%

The percentage cleavage results are also illustrated in the bar graph ofFIG. 13, in which the darker bars represent the results of Run 1, andthe lighter bars represent the results of Run 2.

FIG. 14 are fluorescence images obtained for Run 2, using an EVOSfluorescent microscope with an RFP light cube to detect the labeledtracrRNA. As can be seen in the images, the HPRT and the non-targetingRNPs successfully transfected into the cells under experimentalconditions while the negative control conditions resulted in very lowuptake.

We claim:
 1. A method for transfecting host cells, comprising contactinghost cells in a reservoir containing, in a host-cell compatible fluidmedium, microbubble-antibody conjugates and an exogenous material to betransfected into the host cells, and sonoporating the host cell-dilutionmixture so provided by irradiating the reservoir with acoustic radiationgenerated by two transducers operating in concert but at differentfrequencies, wherein one of the transducers comprises an annulartransducer operably mounted around and enclosing a standard transducer.2. The method of claim 1, wherein the annular transducer operates at afrequency in the range of about 1 MHz to about 2.5 MHz.
 3. The method ofclaim 2, wherein the standard transducer operates at a frequency in therange of about 6 MHz to about 20 MHz.
 4. The method of claim 1, whereinone of the two transducers primarily functions to supply the acousticenergy for sonoporation and the other transducer delivers acousticenergy to enable acoustic ejection of sonoporated cells from the fluidmedium.
 5. The method of claim 1, wherein one of the two transducersprimarily functions to supply the acoustic energy for sonoporation andthe other transducer delivers acoustic energy to change the relativeposition of the microbubbles with respect to the host cells.
 6. Themethod of claim 5, wherein the relative position of the microbubbleswith respect to the host cells does is changed to bring the microbubblesand the host cells in proximity of each other to facilitatesonoporation.
 7. The method of claim 4, wherein the microbubbles and thehost cells are positioned in proximity of each other in a region ofacoustic intensity effective to cause sonoporation.
 8. The method ofclaim 1, wherein the reservoir is contained within a plurality ofreservoirs each comprising additional host cells, microbubble-antibodyconjugates, an exogenous material to be transfected into the host cells,and a host cell-compatible fluid medium.
 9. The method of claim 8,further comprising repeating the method for the additional reservoirs totransfect the additional host cells therein.
 10. The method of claim 9,wherein the method is repeated with a reservoir-to-reservoir transitiontime of at most about 0.5 seconds.
 11. The method of claim 10, whereinthe reservoir-to-reservoir transition time is at most about 0.1 seconds.12. The method of claim 11, wherein the reservoir-to-reservoirtransition time is at most about 0.001 seconds.
 13. The method of claim8, wherein the plurality of reservoirs comprises 96 reservoirs, 384reservoirs, 1536 reservoirs, 3456 reservoirs, or more than 3456reservoirs.
 14. The method of claim 13, wherein the reservoirs arearranged in an array.
 15. The method of claim 14, wherein the reservoirsare contained within a substrate comprising an integrated multiplereservoir unit.
 16. The method of claim 15, wherein the integratedmultiple reservoir unit comprises a well plate.
 17. In a method fortransfecting host cells using sonoporation, the improvement comprisingevaluating transfection efficiency following a sonoporation event,adjusting at least one sonoporation parameter as a candidate solution toimprove transfection efficiency, conducting an additional sonoporationevent, and evaluating transfection efficiency of the additionalsonoporation event to assess effectiveness of the candidate solution.18. The method of claim 17, further comprising changing sonoporationparameters based on the transfection efficiency of the additionalsonoporation event.
 19. The method of claim 17, wherein the sonoporationparameter is selected from at least one of acoustic intensity,transducer output frequency, and toneburst profile.